The AKT signaling pathway is frequently hyperactivated by a variety of mechanisms in a wide range of human cancers, including melanoma, breast, lung, prostate, and ovarian tumors (see Vivanco I and Sawyers C L (2002) Nat Rev Cancer. 2(7):489-501; Scheid M P and Woodgett J R (2001) J Mammary Gland Biol Neoplasia. 6(1):83-99). In tumor cells, the AKT protein kinase activity can be elevated by amplification and overexpression of the AKT2 gene, or by increased production of phosphatidylinositol (3, 4, 5) trisphosphate (PIP3), which activates AKT by recruitment to the plasma membrane. In normal phosphoinositide metabolism, phosphatidylinositol (3, 4) bisphosphate (PIP2) is phosphorylated by phosphatidylinositol 3-kinase (PI3K) to generate PIP3, and PIP3 is dephosphorylated back to PIP2 by the lipid phosphatase PTEN. PIP3 levels in tumors can be enhanced by amplification and overexpression of PI3K, or by hyperactivation of the PI3K activator IGF receptor. Most commonly, however, PIP3 levels in tumor cells are elevated by mutation or deletion of the PTEN tumor suppressor, at rates as high as 40-50% of prostate cancers. The AKT pathway promotes tumor progression by enhancing cell proliferation, growth, survival, and motility, and by suppressing apoptosis. These effects are mediated by several AKT substrates, including the related transcription factors FKHR and AFX, for which phosphorylation by AKT mediates nuclear export.
All of the major AKT pathway components have structural and functional orthologs in C. elegans that function in dauer larva formation (see Wolkow C A et al. (2002) J Biol Chem 277(51):49591-7; Paradis S et al (1999) Genes Dev. 13(11):1438-52). Normally, environmental cues of low food (bacteria) levels, high dauer pheromone concentration, and high temperature trigger a developmental decision that signals alternative differentiation pathways in all tissues and entry into a diapause (dauer) arrest. Inactivating mutations or RNAi of the AKT orthologs akt-1 and akt-2, or the PI3K ortholog age-1, or the IGP receptor daf-2, produce a dauer-constitutive (Daf-c) phenotype; in which animals form dauers even under environmental conditions that normally induce development to adulthood. Conversely, inactivating mutations in the PTEN ortholog daf-18, or the FKHR and AFX ortholog daf-16, generate a dauer-defective (Daf-d) phenotype and prevent dauer formation regardless of environmental conditions. A daf-18 deletion mutant fully suppresses the Daf-c phenotype of age-1 and daf-2 mutations (Gil E B et al (1999) Proc Natl Acad Sci USA. 96(6):2925-30; Mihaylova V T et al (1999) Proc Natl Acad Sci USA. 96(13):7427-32), and we have identified two loss-of-function mutations that allow non-dauer development of the heat-sensitive daf-2 (1370) allele at the non-permissive temperature of 25° C. daf-18 (ep496) is a nonsense mutation at amino acid E455, and daf-18 (ep497) is a missense mutation predicted to cause the amino acid substitution G80D. The Daf-d phenotype of double mutants daf-18 (ep496); daf-2 (e1370) and daf-18 (ep497); and daf-2 (e1370) can be reverted to a Daf-c phenotype by RNAi of akt-1 or age-1, indicating that the double mutants display increased AKT signaling.
Microtubules have a central role in the regulation cell shape and polarity during differentiation, chromosome partitioning at mitosis, and intracellular transport. Microtubules undergo rearrangements involving rapid transitions between stable and dynamic states during these processes. Microtubule affinity regulating kinases (MARK) are a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption (Drewes, G., et al. (1997) Cell 89: 297-308). Mammalian SNF1 like kinase (SNF1LK) is a serine/threonine kinase similar to Snf1 protein kinase, of S. cerevisiae, which is involved in the response to nutritional stress.
The ability to manipulate the genomes of model organisms such as C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Dulubova I, et al, J Neurochem April 2001; 77(1):229-38; Cai T, et al., Diabetolbgia January 2001; 44(1):81-8; Pasquinelli A E, et al., Nature. Nov. 2, 2000; 408(6808):37-8; Ivanov I P, et al., EMBO J Apr. 17, 2000; 19(8): 1907-17; Vajo Z et al., Mamm Genome October 1999; 10(10):1000-4). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as PTEN, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.